1. Field of the Invention
The present invention relates to a biochemical examination method for examining a state of a biochemical reaction.
2. Description of the Related Art
In recent years, for “determination of arrangement by hybridization” (SBH), several methods of an arrayed hybridization reaction have been developed. Examples include an SBH using a method of a stepwise hybridization of different oligonucleotide probes and an array of DNA samples arranged on a film in a lattice form.
Moreover, a term “genosensor” has heretofore referred to a method in which oligonucleotide is bonded as a recognition element of a target nucleic acid arrangement and a complementary arrangement on the surface of a two-dimensional array. Furthermore, a concept of genosensor includes a fine processing apparatus in which hybridization can quickly be detected and a fine electronic component is present in each test portion.
With respect to the two-dimensional array, in recent years, a flow through genosensor has been provided as follows. In the genosensor, the nucleic acid recognition element is immobilized in densely charged holes or channels arranged in a spotted form over a wafer of a solid support material. A known fine processing technique can be utilized in manufacturing glass and porous silicon of a micro channel or a nano-channel useful as the support wafer. For the flow through genosensor, various known detection methods are used which include finely processed optical and electronic engineering detection components, film, charge bonding element array, camera system and phosphorescence storage technique. The following advantages are obtained from the flow through apparatus as compared with a known plane surface design.
(1) Since a surface area enormously increases, detection sensitivity is improved.
(2) A time required for the hybridization reaction is reduced (a time required for detecting the probe with an average target molecule bonded to the surface thereof is reduced to several milliseconds from several hours), the hybridization is speeded up, and error-pairing (error-pass) identification can be made in both normal reaction and reverse reaction.
(3) Since a solution can gradually flow through the porous wafer, a dilute nucleic acid solution can be analyzed.
(4) Since a droplet of a probe solution on a plane surface exposed to the atmosphere is prevented from quickly drying, chemical bond of a probe molecule to the surface in each separate region is promoted.
The flow through apparatus having the above-described advantages will be referred to as a three-dimensional array. PCT National Publication No. 1997-504864 is cited as a prior art relating to the three-dimensional array, and FIG. 27 is used to briefly describe the constitution and action of the array.
FIG. 27 is a diagram showing a tapered sample well array constituting the three-dimensional array. In FIG. 27, a plurality of tapered holes 102 are formed in a porous glass wafer 101, and tapered wells 103 are buried in the respective holes 102. The tapered well 103 includes a channel 104 having a diameter of 0.1 to 10 μm and constituting a bonding region of a bio-molecule immobilized thereto in the bottom of the well. As shown in FIG. 27, each channel 104 has a large number of micro through holes 105. The well array is used, and detection is performed in the following steps.
(1) A solution containing 4 ml of 3-glycidoxypropyl-trimetoxysilane, 12 ml of xylene, and 0.5 ml of N,N-di-isopropyl ethyl amine (Hunig base) is passed through the holes of the wafer. Subsequently, the wafer is immersed in the solution at 80° C. for five hours, flushed with tetrahydrofuran, dried at 80° C., and thereby formed into epoxysilane-derivative glass.
(2) A plurality of oligonucleotide probes having 5′- or 3′-alkylamine (introduced during chemical synthesis) are dissolved in water at a ratio of 10 μM–50 μM, and a micro amount of the solution is pipetted in the porous glass wafer 101 (silica wafer). The wafer is reacted at 65° C. overnight, the surface of the wafer is simply flushed with water at 65° C., then with 10 mM triethylamine, and a non-reacted epoxy group is removed from the surface. Subsequently, the wafer is again flushed with water at 65° C. and air-dried, so that amine-derivative oligonucleotide is bonded to epoxysilane-derivative glass.
(3) By polymerase chain reaction in which [32P] nucleotide is taken into a product during amplification, or 5′-labeling of the amplified product using gamma-32P[ATP]+polynucleotide kinase, a target DNA (analysis material) is prepared. Not-taken markers are removed by Centricon filtering. Preferably, with 5′-biotin labeling of one PCR fragment, one chain can be prepared by streptoavidin affinity chromatography. The target DNA is dissolved in a hybridization buffer solution (50 mM tris-HCl, pH 8, 2 mMEDTA, 3.3M tetramethyl ammonium chloride) having a concentration of at least 5 nM (5 fmol/μl) and a specific activity of at least 5,000 cpm/fmol. The PCR fragment having several hundreds base length is suitable for the hybridization with oligonucleotide linked to the surface having at least octameter length.
(4) The target DNA sample is passed into the porous region of a chip, and incubated at 6° C. for 5 to 15 minutes, and the hybridization is performed. Subsequently, the hybridization solution is allowed to flow through the porous chip at 18° C. similarly for 5 to 15 minutes and the chip is washed. As another method, instead of tetramethyl ammonium chloride, the buffer solution containing 1MKCL, NaCl or 5.2M betaine can be used to perform the hybridization.
(5) A CCD genosensor apparatus is used to perform detection and quantitative determination of a hybridization intensity. The CCD genosensor apparatus having high resolution and sensitivity is used, and prepared for chemical luminescent, fluorescence or radioactivity marker.
In conventional arts including the above-described prior art, when each biochemical reaction state in a probe array element is detected as luminescent intensity by the same area sensor such as CCD or a line sensor, all the probe array elements need to be uniformly irradiated. This is because a luminescent state of a luminescent molecule depends on the intensity of excitation light.
It is easily considered that the intensity of the excitation light is monitored and a detected value is corrected in a space distribution of the intensity. However, even in this case, a relation between the intensity of the excitation light and the luminescent intensity is complicated, and the relation changes with the density of the luminescent molecule, temperature, time, and the like. Therefore, there is a disadvantage that each biochemical reaction state in the probe array element cannot exactly be examined.
Moreover, a noise of excitation light or a noise by a pipetting error of the probe cannot sufficiently be removed. Furthermore, the luminescent property of the luminescent molecule is not considered, and therefore there is a disadvantage that each reaction state cannot exactly be examined.